Methodology A novel method for purifying bluetongue virus with high purity by co-immunoprecipitation with agarose protein A Song Zhen1, Dong Changyuan*1, Wang Lulu1, Chen Dong-E2, Bi Gu
Trang 1Open Access
M E T H O D O L O G Y
© 2010 Zhen et al; licensee BioMed Central Ltd This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Methodology
A novel method for purifying bluetongue virus
with high purity by co-immunoprecipitation with agarose protein A
Song Zhen1, Dong Changyuan*1, Wang Lulu1, Chen Dong-E2, Bi Guoming1, Dai Ming1 and Liu Jun1
Abstract
Background: Bluetongue virus (BTV) is an icosahedral non-enveloped virus within the genus Orbivirus of Reoviridae
and exists as 24 distinct serotypes BTV can infect all ruminant species and causes severe sickness in sheep Recently, it was reported that BTV can infect some human cancer cells selectively Because of the important oncolysis of this virus,
we developed a novel purifying method for large-scale production The purifying logic is simple, which is picking out all the components unwanted and the left is what we want The process can be summarized in 4 steps: centrifugation, pulling down cell debrises and soluble proteins by co-immunoprecipitation with agarose Protein A, dialysis and filtration sterilization after concentration
Results: The result of transmission electron microscope (TEM) observation showed that the sample of purified virus
has a very clear background and the virions still kept intact The result of 50% tissue culture infective dose (TCID50) assay showed that the bioactivity of purified virus is relatively high
Conclusions: This method can purify BTV-10 with high quality and high biological activity on large-scale production It
also can be used for purifying other BTV serotypes
Background
Bluetongue virus (BTV) is an icosahedral non-enveloped
virus within the genus Orbivirus of Reoviridae [1] and
exists as 24 distinct serotypes [2] BTV can infect
rumi-nant species mainly through the biting Culicoides species
[3] Particularly, it usually causes severe bluetongue
dis-eases (BT) only in sheep and some species of deer Over
the past century, BTV has never shown infectivity on
Homosapiens, and no normal human being cells have
been successfully infected by BTV [4]
The genome of BTV contains ten linear segments of
double-stranded RNA, and each of them codes one of the
viral proteins The virion of BTV has two protein shells
with about 850-Å-diameter The innermost shell is
com-posed of 120 copies of protein VP3 (about 103 KD),
which encloses three proteins (VP1, VP4 and VP6) and 10
segments of dsRNA genome, while the outer shell
con-sists of protein VP2 (111 KD) and protein VP5 (about 59
KD) The two shells are linked by 780 copies of protein VP7 (about 38 KD) [5]
Protein VP2, coded by L2 segment, is the major cell adhesion protein [6-8] and the most variable protein in BTV It can induce neutralizing antibody in the infected host On the basis of the antigenicity of protein VP2, all of the 24 serotypes of BTV can be distinguished [9,10] Besides, Protein VP5 helps to control the serotype of BTV [11]
Recently, BTV-10 has been reported that it can infect some human cancer cells selectively The viral dose-dependent cytopathic effect (CPE) can be effectively induced by both virion amplification and virus-induced apoptosis on human liver carcinoma cell line (Hep-3B) and human lung carcinoma cell line (A549), while no visi-ble CPE could be observed or detected in primary human embryo lung fibroblast cell (HEL) even after 5th day post-infection [4] Another study about an unserotyped BTV, which was isolated from Xiangfan, Hubei province of
cause apoptosis on Hep-3B cells and paraptosis on A549 cells [12] Further more, in the in vivo test this strain can
* Correspondence: labofmvc@whu.edu.cn
1 Lab of Molecular Virus & Cancer, State Key Laboratory of Virology, Wuhan
University School of Basic Medicine, Wuhan University, Wuhan 430071, China
Full list of author information is available at the end of the article
Trang 2make MA782-induced subcutaneously grown breast
ade-nocarcinoma significantly regressed in mice model [13]
There are many reports about the methods for BTV
purification, such as CsCl or saccharose density-gradient
centrifugation But these methods could hardly produce
large amount of BTV virions and make BTV somewhat
degraded [14-16] We developed an effective method for
high-throughput purifying BTV with high bioactivity,
which is very helpful for the study of BTV anti-tumor
effect since such studies need plenty of BTV with high
bioactivity
Results
Purity and integrity of purified BTV-10 by transmission
electron microscopy (TEM)
The photographs from TEM observation showed both
unpurified (Fig 1) and purified (Fig 2) negatively stained
samples In the photograph of unpurified virus, limited
amount of virions can be observed and they were
sur-rounded with a mass of cell debris (Fig 1A &1B) On the
contrary, the photograph of purified sample revealed the
purity and integrity of virus In fact, the virions can be
clearly observed in a clean background; meanwhile, the
purified viral particles were still kept intact (Fig 2A &2B)
The biological infectivity of purified and unpurified BTV was compared by the infectivity curvilinear trend and a TCID50 assay Fig 3 showed the process of 50% cells exhibiting CPE as the infected time goes on according to the number of wells, and as the curvilinear trend shown the infectivity of purified virus is similar to the unpurified virus The TCID50 was calculated at the seventh day when the cells being infected The TCID50 of unpurified BTV
TCID50/ml under the same conditions
Discussion
Nowadays, the research on anti-tumor mediated by virus
is a hot field Several viruses have been reported that they can infect tumor cell lines obviously, such as Reovirus [17] and Newcastle disease virus [18] Over the past
cen-Figure 1 Transmission electron microscopy pictures of the
unpu-rified The sample was a direct collection of the virus culture on Vero
cells Picture A was photographed by digital camera and the virions
were marketed by arrows Picture B was photographed by film and the
virions were marketed by arrows In picture B, an empty shell was
ob-served Cell debris was also clearly observed in the two pictures.
Figure 2 Transmission electron microscopy pictures of the puri-fied Picture A was photographed by digital camera and the virions
were marketed by arrows The two-layer structure of BTV virion was perfectly shown in picture A Picture B was photographed by film and the virions were marketed by arrows In picture B, an uncoated BTV vi-rion was observed and was marked by arrow.
Figure 3 The activity of purified and unpurified virus The chart
graph shows the process of 50% cells exhibiting CPE as the infected time goes on according to the number of wells The unpurified virus was used as a criterion to measure whether the purified virus was still active enough As the infecting days went by, the number of wells ap-peared 50% CPE was increasing The curvilinear trend of purified virus
is in accordance with unpurified virus, and the wells exhibiting 50% CPE in purified virus per day is slightly less than that of unpurified virus.
Trang 3tury, BTV has never been reported infectivity on
Homo-sapiens, and no normal human being cells have been
infected by this virus We have studied BTV on
anti-tumor for a long time and found that it can infect A549
and Hep-3B cell lines in vitro [4] and MA782 cell line in
vivo However, further study needs a large amount of
viri-ons with high biological activities, and current virus
puri-fication method can not meet our needs of researching
This study introduced a novel method for purifying
BTV-10 by cell debris co-immunoprecipitation with
aga-rose Protein A The logic is pulling out all the
compo-nents unwanted, and then the left is what we want The
whole process can be summarized as three purification
steps as well as one condensation and filtration
steriliza-tion step The culture including Vero cells and the
nutri-ent medium, which is used to proliferate BTV, were used
as antigen to inoculate experimental rabbits in order to
acquire polyclonal antibody These antibodies containing
various components were purified twice and then
absorbed by protein A formerly bounded on agarose At
the same time, the culture mixture, containing lysed Vero
cells, was centrifuged so as to remove some cell apparatus
and big cell fragments This step is very important and
can be regarded as the first step of purification Then the
virus supernatant was mixed with the antibody-protein
A-agarose complex In this second step, molecules and
cell pieces, which have antigenicity, have been pulled
down mostly, whereas BTV virions still kept in the
super-natant But the second step is still not enough In the
supernatant, there were still a mass of molecules with no
immunogenicity and those molecules that having
antige-nicity but did not induce immune response So dialysis
was needed, as the third step, to remove these
ingredi-ents Thanks to the diameter of BTV particles is 850Å, it
is safe enough to use dialysis tubing with 300 KD filtering,
which can effectively remove all of the left molecules
After this third step, there were just BTV virions and
small amount of microorganisms left And the final step
was the condensation of the virus solution by
Polyethyl-ene Glycol (PEG-20000) and sterilization filtration by 0.1
μ membrane filter
The photographs about TEM observation on purified
virus showed clearly that the purified viral particles were
intact with a clear and clean background, while the
unpu-rified sample's photographs showed few virions that were
surrounded by a large amount of cell debris This
phe-nomenon means the virions were successfully separated
from a mass of castoff
Biological activity of purified viruses was tested by a
TCID50 assay in Vero cells Compared with unpurified
virus, the TCID50/ml of the purified fell slightly This
fall-ing may be caused by filtration sterilization Considerfall-ing
that mycoplasma, which is a kind of potential pollutant in
purification process, can penetrate 0.22 μ filter
mem-brane, and the diameter of filter membrane we used for sterilization is 0.1 μ, which is very close to the diameter of
a BTV particle, so as to avoid the purified virus contami-nated by mycoplasma as much as possible Unfortunately, this step may cause virions loss
The purification process completely focuses on how to remove impurities with no regard to BTV serotypes, which is just like centrifugation method but can avoid physical injury on virions effectively Thus it can be used for all BTV serotype's purification and we have purified several serotypes of BTV with this method (data not shown)
Conclusions
After the above analysis based on the experiment, a new BTV purification method with high quality and high bio-logical activity was established, and this may significantly push forward the research of BTV on anti-tumor and blu-etongue sickness
Methods
BTV-10 proliferation
BTV-10 and other serotype strains were acquired from China Animal Health and Epidemiology Center Vero cell line was cultured in MEM (Gibco, USA) with 5% fetal bovine serum at 37°C, 5% CO2 When cells formed monolayer, culture medium was discarded and cells were washed by Phosphate Buffered Saline (PBS) (50 mM PB,
100 mM NaCl, 1 mM EDTA, pH7.4) twice Subsequently, the monolayer of Vero cells were infected with 0.5 ml BTV-10 suspension (105 TCID50/ml) and incubated at 37°C for 2 hours After that, 6-10 ml MEM with 10% fetal bovine serum was added, and cells were further incu-bated at 37°C When 90% cells appeared CPE, the culture mixture was subjected to three rapid freeze/thaw cycles
by putting it into liquid nitrogen and 37°C water bath Then the virus-cell mixture (15 ml) was centrifuged at
5000 g for 10 minutes at 4°C and the supernatant was col-lected We took out 5 ml supernatant (virion-cell debris) and preserved it at -20°C as control virus The left virion-cell debris suspension was centrifuged at 12,000 × g for 10 minutes at 4°C And then the supernatant was harvested and stored at -20°C The uninfected cells were cultured in the same conditions as the control, which were used for producing polyclonal antibody either
Preparation of Antibodies
The Vero cell-culture mixture was subjected to three rapid freeze/thaw cycles by putting it into liquid nitrogen and 37°C water bath Following ultrasonication for three 1-minute cycles in ice-bath, the cell debris suspension was centrifuged at 5000 × g for 10 minutes at 4°C Subse-quently, the supernatant was used as the antigen injected into experimental rabbits to produce polyclonal antibody
Trang 4The first inoculation was performed with 0.2 ml
anti-gen and Freund's incomplete adjuvant (FIA) mixture and
BCG vaccine (Bacillus Calmette-Guerin) to final
concen-tration of 10 mg/ml 2 weeks later, the second inoculation
was performed the same dose Then after 2 weeks,
anti-gen without Freund's adjuvant was inoculated 3 days
con-tinuously (once a day) with 0.1 ml, 0.2 ml and 0.3 ml
respectively as consolidating immunization One week
later, the blood was collected from experimental rabbits'
heart and then the serum was harvested The first
purifi-cation of the antibodies was precipitation by saturated
(NH4)2SO4 The sediment was resolved in 0.9% NaCl
solution and was dialyzed by PBS at 4°C and the dialysis
buffer was changed every 6 hours until the (NH4)2SO4
was completely removed The trace amount of (NH4)2SO4
was checked by BaCl2 solution Then the polyclonal
anti-body was further purified by Montage® Antibody
Purifica-tion K with PROSEP-A media
Purification of BTV-10 by Reverse Co-immunoprecipitation
with Agarose Protein A
2000 μl antibodies (2 mg/ml) were mixed with 3 ml
aga-rose Protein A completely and incubated at 4°C
over-night Then the mixture was centrifuged at 1000 × g for
10 minutes at 4°C The supernatant was discarded while
the deposit was collected and put into 10 ml unpurified
BTV-10 supernatant The deposit, containing antibodies
and agarose Protein A, and BTV-10 suspension was
incu-bated at 4°C overnight After incubation, the mixture was
centrifuged at 1000 × g for 10 minutes at 4°C
Purification of BTV-10 by dialysis
Following the preceding step, the supernatant was
trans-ferred into dialysis tubing with 300 KD molecular weight
cutoff (MWCO) and was dialyzed by PBS at 4°C for 6
hours, which is one of the cell friendly buffers Then, the
dialyzed solution is concentrated by PEG-20000 and
desalted by dialyzing at the same buffer again This
pro-gram was repeated 4 times
Transmission Electron microscopy assay
The purified BTV suspension was sterilized by filtration
using 0.1 μ membrane filter firstly Then both of the
puri-fied and unpuripuri-fied virus suspension were negatively
stained with 2% phosphotungstic acid They were
observed by electron microscope to compare the purity
and integrity of viruses
Identification of biological activity of purified viruses
BTV TCID50 assay was used to test biological activity of
BTV in Vero cells Vero cells were cultured in two plates
of 96 wells When Vero cells formed monolayer, in one
plate, they were infected with the purified virus
suspen-sion formerly sterilized by filtration; in the other plate,
the cells were infected with the unpurified virus suspen-sion Both purified and unpurified virus suspensions were diluted 10 times for six serial dilutions arrange from 10-3
-10-8 Each dilution was added to 12 wells with 100 μl/well Meanwhile 24 wells (F line and G line) of cells of each plate were cultured as the control group The two plates were placed at 37°C with 5% CO2 and CPE was observed everyday for 7 days And TCID50 was calculated using Karber formula
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
SZ carried out Preparation of Antibodies, dialysis, concentration, drafted the manuscript and participated in TEM observation DCY as the corresponding author designed the idea of the method and participated in TEM observation CDE and WLL carried out the biological activity test, and WLL participated in revising the manuscript Bi Guoming carried out the Reverse Co-immunopre-cipitation; DM and LJ carried out BTV proliferation All authors have read and approved the final manuscript.
Acknowledgements
This study was supported by National Natural Science Fund (No 30772519).
Author Details
1 Lab of Molecular Virus & Cancer, State Key Laboratory of Virology, Wuhan University School of Basic Medicine, Wuhan University, Wuhan 430071, China and 2 Dept of Epidemiology & Health Statistics, Wuhan University School of Public Hygienics, Wuhan University, Wuhan 430071, China
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Received: 16 February 2010 Accepted: 13 June 2010 Published: 13 June 2010
This article is available from: http://www.virologyj.com/content/7/1/126
© 2010 Zhen et al; licensee BioMed Central Ltd
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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doi: 10.1186/1743-422X-7-126
Cite this article as: Zhen et al., A novel method for purifying bluetongue
virus with high purity by co-immunoprecipitation with agarose protein A
Virology Journal 2010, 7:126